Jensen D E, Kelly R C, von Hippel P H
J Biol Chem. 1976 Nov 25;251(22):7215-28.
Bacteriophage T4-coded gene 32-protein is an essential component of the T4 replication and recombination systems. Alberts and co-workers (Alberts, B.M., Amodio, F.J., Jenkins, M., Gutmann, E.D., and Ferris, F.L. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 289-305) have shown that the major physiological activity of the protein involves preferential and cooperative binding to single-stranded DNA. In this paper, the physiochemical parameters characterizing this "melting" protein system are quantitatively determined. Boundary sedimentation velocity experiments are used to measure the interaction of gene 32-protein with native DNA. The binding is shown to be non-cooperative and involves an overlapping site size (nh) of approximately 10 nucleotide residues (or approximately 5 nucleotide pairs). In analogy with the ribonuclease results (Jensen, D.E., and von Hippel, P.H. (1976) J. Biol. Chem. 251, 7198-7214), the logarithm of the association constant (Kh) is found to be linerarly related to log [Na+]. The binding of gene 32-protein to denatured (single-stranded) DNA involves appreciable distortion of the polynucleotide backbone from the unliganded conformation; binding totally unstacks the bases of both ribose- and deoxyribose-containing polynucleotides at 10 degrees, and results in a hyperchromic change exceeding that which can be induced by heating. This hyperchromism induced in poly(dA) on binding gene 32-protein under low salt (tight binding) conditions is used to determine a value of nc (the single-stranded DNA site size) of approximately 6.7 nucldotide residues per protein. In addition, gene 32-protein binding to single-stranded polynucleotide induces an unusual circular dichroic spectrum characterized principally by a marked decrease in the magnitude of the positive CD band centered at approximately 265 nm. This spectral change is attributed to significant uncoupling of the transition moments of the vicinal bases of the single-stranded polynucleotide on gene 32-protein binding, in accord with the ultraviolet hyperchromism observed. Binding of gene 32-protein to double helical DNA has virtually no effect on the spectral properties of this conformation...
噬菌体T4编码的基因32蛋白是T4复制和重组系统的重要组成部分。阿尔伯茨及其同事（阿尔伯茨，B.M.，阿莫迪奥，F.J.，詹金斯，M.，古特曼，E.D.，和费里斯，F.L.（1968年）《冷泉港定量生物学研讨会》33卷，289 - 305页）已经表明，该蛋白的主要生理活性涉及与单链DNA的优先结合和协同结合。在本文中，定量测定了表征这种“解链”蛋白系统的物理化学参数。采用边界沉降速度实验来测量基因32蛋白与天然DNA的相互作用。结果表明这种结合是非协同的，涉及的重叠位点大小（nh）约为10个核苷酸残基（或约5个核苷酸对）。与核糖核酸酶的结果（詹森，D.E.，和冯·希佩尔，P.H.（1976年）《生物化学杂志》251卷，7198 - 7214页）类似，发现缔合常数（Kh）的对数与log [Na⁺]呈线性关系。基因32蛋白与变性（单链）DNA的结合涉及多核苷酸主链从无配体构象的明显扭曲；在10℃时，结合使含核糖和脱氧核糖的多核苷酸的碱基完全解堆积，并导致增色变化超过加热所能诱导的程度。在低盐（紧密结合）条件下结合基因32蛋白时，多聚（dA）中诱导的这种增色作用被用于确定每个蛋白的nc（单链DNA位点大小）值约为6.7个核苷酸残基。此外，基因32蛋白与单链多核苷酸的结合诱导出一种不寻常的圆二色光谱，其主要特征是在约265nm处的正CD带的幅度显著降低。这种光谱变化归因于基因32蛋白结合时单链多核苷酸邻位碱基跃迁矩的显著解耦，这与观察到的紫外增色现象一致。基因32蛋白与双螺旋DNA的结合对这种构象的光谱性质几乎没有影响……
